Académique Documents
Professionnel Documents
Culture Documents
Objectives
1. Understand the terms enrichment, selective, and differential
medium.
2. Understand the difference between culturable counts and direct
counts.
3. Understand the difference between MPN and culturable counts
4. Given a soil or water sample be able to describe a dilution
scheme.
5. Be able to convert soil wet weight results to soil dry weight results.
Cultural Methods
Bacterial numbers can be determined by direct counts or by culturable
counts.
Culturable counts
Plate counts are performed using dilution and plating
The choice of plating medium is important
- enrichment media allow one to enrich for certain populations
- selective media contain components that select against certain
populations
- differential media contain components that allow certain populations to
stand out on the plating medium
low pH
rose bengal (dye)
streptomycin
neomycin
What antibiotic would you add to a general medium to select for bacteria ?
Saturation
status
AODC (cells/g)
PTYG agar
5% PTYG agar
SSA
0.3
U topsoil
1.6
U vadose
3.8
7.4
7.9
Consolidated
U = unsaturated
S = saturated
Consolidated = mostly nontransmissive zone beneath aquifer
Dilution series
0.1 ml
Dilution = 10-4
10-5
10-6
saline
95 ml
dilution
10-1
1 ml
9 ml
10-2
1 ml
9 ml
10-3
1 ml
9 ml
10-4
1 ml
9 ml
10-5
Plate 0.1 ml
CFU =
1
x number of colonies
dilution factor
10-6
Dilution =
10-4
10-5
10-6
Always count plates with 30 to 300 colonies. Less is not statistical and more
is too numerous to count.
For comparison with other samples, express CFUs in terms of the dry
weight of the soil.
To determine dry weight, weigh a sample, dry it in an oven, reweigh it
and then calculate the % moisture.
If you weigh 1 g of soil, dry it and find it then weighs 0.8 g, it has a 20%
moisture content.
Moisture content = moist wt - dry wt
dry wt
0.2 = 1 g dry wt
dry wt
dry wt =
0.833 g
Direct counts
Give an estimate of the total number of bacteria in a sample. This is
useful for a variety of habitats and has no specific bias for particular
genera. On the other hand, it is hard to distinguish living from dead
cells.
Direct counts are performed using the following techniques:
fluorescent staining and epifluorescence microscopy.
acridine orange (AODC)
46-diamino-2-phenylindole (DAPI)
fluorescein isothiocyanate (FITC)
Molecular probes
fluorescent antibodies
electron microscopy (virus)
particle counter (coulter counter, flow cytometer)
Sample
Soil
Direct counts
Cells/g
Soil
Culturable counts
CFU/g
Marine water
Direct counts
Cells/ml
Marine water
Culturable counts
CFU/ml
5.0 x 108
3.1 x 107
2.2 x 103
1.3 x 101
1.1 x 109
6.2 x 107
8.2 x 104
7.6 x 102
2.0 x 109
1.7 x 108
1.3 x 106
2.1 x 104
20
15
10
r2 = 0.97
5
0
0
20
40
60
80
100
120
Culturing viruses
Living cells are required to detect virus replication whether they are animal or
bacterial cells. Usually a lawn or a confluent layer of cells is prepared. The
sample (presumably containing virus) is added. The plates are incubated
and evaluated for:
CPE (cytopathogenic effects)
PFU (plaque forming units)
CPE
Infected culture
Normal culture